UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes.

The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5'-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin.

Evaluation of urinary cotinine immunoassay test strips used to assess smoking status.

INTRODUCTION: Validation of smoking status is a vital component for assessing self-reported smoking status. This study examined commercially available urinary cotinine immunoassay strips to evaluate their effectiveness and their reliability in validating self-reported tobacco usage. METHODS: This study compared cotinine values from urinary test strips and liquid chromatography-mass spectrometry (LC-MS/MS) obtained from 100 self-reported nonsmokers and 201 self-reported smokers. RESULTS: The analyzed test strips, with 100 ng/ml as the cutoff value, have 91% (84%-96% CI) specificity and 92% (86%-95% CI) sensitivity. The test strips successfully identified nonsmokers from smokers with excellent reproducibility. However, self-reported smokers have significantly different (p < .001) test strip results when compared with the expected LC-MS/MS values. CONCLUSIONS: Overall, the test strip demonstrated that it discriminates nonsmokers from smokers and is an adept alternative to chromatographic methods.

The genotoxicity of mainstream and sidestream marijuana and tobacco smoke condensates.

While the prevalence of tobacco use has decreased in Canada over the past decade, that of marijuana use has increased, particularly among youth. However, the risks of adverse health effects from marijuana smoke exposure, specifically as compared to tobacco, are currently not well understood. The objectives of this study were to evaluate the relative ability of matched marijuana and tobacco condensates to induce (geno)toxic responses in three in vitro test systems. This study provides comparative data for matched sidestream and mainstream condensates, as well as condensates prepared under both a standard and an extreme smoking regime designed to mimic marijuana smoking habits. The results indicate that tobacco and marijuana smoke differ substantially in terms of their cytotoxicity, Salmonella mutagenicity, and ability to induce chromosomal damage (i.e., micronucleus formation). Specifically, the marijuana condensates were all found to be more cytotoxic and more mutagenic in the presence of S9 than the matched tobacco condensates. In contrast, the tobacco condensates appeared to induce cytogenetic damage in a concentration-dependent manner, whereas the matched marijuana condensates did not. In addition, when corrected for total particulate matter yield, little difference was observed in the mutagenic activity of samples smoked under the extreme vs the standard regime for both tobacco and marijuana condensates.

The accuracy of self-reported smoking: a systematic review of the relationship between self-reported and cotinine-assessed smoking status.

INTRODUCTION: Smoking is a leading cause of premature mortality and preventable morbidity. Surveillance is most often based on self-reported data, but studies have shown that self-reports tend to underestimate smoking status. METHODS: This study systematically reviewed the literature to measure the concordance between self-reported smoking status and smoking status determined through measures of cotinine in biological fluids. Four electronic databases were searched to identify observational and experimental studies on adult populations over the age of 18 years. RESULTS: Searching identified 67 studies that met the eligibility criteria and examined the relationship between self-reported smoking and smoking confirmed by cotinine measurement. Overall, the data show trends of underestimation when smoking prevalence is based on self-report and varying sensitivity levels for self-reported estimates depending on the population studied and the medium in which the biological sample is measured. Sensitivity values were consistently higher when cotinine was measured in saliva instead of urine or blood. Meta-analysis was not appropriate because of the substantial heterogeneity among the cutpoints used to define smokers and the poor reporting on outcomes of interest. DISCUSSION: Further research in this field would benefit from the standardization of cutpoints to define current smokers and the implementation of standard reporting guidelines to enhance comparability across studies. Accurate estimation of smoking status is important as data from population studies such as those included in this review are used to generate regional and national estimates of smoking status and in turn are used to allocate resources and set health priorities.

A comparison of mainstream and sidestream marijuana and tobacco cigarette smoke produced under two machine smoking conditions.

The chemical composition of tobacco smoke has been extensively examined, and the presence of known and suspected carcinogens in such smoke has contributed to the link between tobacco smoking and adverse health effects. The consumption of marijuana through smoking remains a reality and, among youth, seems to be increasing. There have been only limited examinations of marijuana smoke, including for cannabinoid content and for tar generation. There have not been extensive studies of the chemistry of marijuana smoke, especially in direct comparison to tobacco smoke. In this study, a systematic comparison of the smoke composition of both mainstream and sidestream smoke from marijuana and tobacco cigarettes prepared in the same way and consumed under two sets of smoking conditions, was undertaken. This study examined the suite of chemicals routinely analyzed in tobacco smoke. As expected, the results showed qualitative similarities with some quantitative differences. In this study, ammonia was found in mainstream marijuana smoke at levels up to 20-fold greater than that found in tobacco. Hydrogen cyanide, NO, NO x , and some aromatic amines were found in marijuana smoke at concentrations 3-5 times those found in tobacco smoke. Mainstream marijuana smoke contained selected polycyclic aromatic hydrocarbons (PAHs) at concentrations lower than those found in mainstream tobacco smoke, while the reverse was the case for sidestream smoke, with PAHs present at higher concentrations in marijuana smoke. The confirmation of the presence, in both mainstream and sidestream smoke of marijuana cigarettes, of known carcinogens and other chemicals implicated in respiratory diseases is important information for public health and communication of the risk related to exposure to such materials.

A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures.

The sensitivity of yeast Saccharomyces cerevisiae to DNA damaging agents is better represented when cells are grown in liquid media than on solid plates. However, systematic assessment of several strains that are grown in different conditions is a cumbersome undertaking. We report an assay to determine cell growth based on automatic measurements of optical densities of very small (100 microl) liquid cell cultures. Furthermore, an algorithm was elaborated to analyze large data files obtained from the cell growth curves, which are described by the growth rate--that starts at zero and accelerates to the maximal rate (mu(m))--and by the lag time (lambda). Cell dilution spot test for colony formation on solid media and the growth curve assay were used in parallel to analyze the phenotypes of cells after treatments with three different classes of DNA damaging agents (methyl methanesulfonate, bleomycin, and ultraviolet light). In these experiments the survival of the WT (wild type) and a number of DNA repair-deficient strains were compared. The results show that only the cell growth curve assay could uncover subtle phenotypes when WT cells, or mutant strains that are only weakly affected in DNA repair proficiency, were treated with low doses of cytotoxic compounds. The growth curve assay was also applied to establish whether histone acetyltransferases and deacetylases affect the resistance of yeast cells to UV irradiation. Out of 20 strains tested the sir2delta and rpd3delta cells were found to be more resistant than the WT, while gcn5delta and spt10delta cells were found to be more sensitive. This new protocol is sensitive, provides quantifiable data, offers increased screening capability and speed compared to the colony formation test.

Psoralen photocrosslinking, a tool to study the chromatin structure of RNA polymerase I--transcribed ribosomal genes.

The chromatin structure of RNA polymerase I--transcribed ribosomal DNA (rDNA) is well characterized. In most organisms, i.e., lower eukaryotes, plants, and animals, only a fraction of ribosomal genes are transcriptionally active. At the chromatin level inactive rDNA is assembled into arrays of nucleosomes, whereas transcriptionally active rDNA does not contain canonical nucleosomes. To separate inactive (nucleosomal) and active (non-nucleosomal) rDNA, the technique of psoralen photocrosslinking has been used successfully both in vitro and in vivo. In Saccharomyces cerevisiae, the structure of rDNA chromatin has been particularly well studied during transcription and during DNA replication. Thus, the yeast rDNA locus has become a good model system to study the interplay of all nuclear DNA processes and chromatin. In this review we focused on the studies of chromatin in ribosomal genes and how these results have helped to address the fundamental question: What is the structure of chromatin in the coding regions of genes?